The Novel Tubulin-binding Drug BTO-956 Inhibits R3230Ac Mammary Carcinoma Growth and Angiogenesis in Fischer 344 Rats1
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
2590 Vol. 7, 2590 –2596, August 2001 Clinical Cancer Research
The Novel Tubulin-binding Drug BTO-956 Inhibits R3230Ac
Mammary Carcinoma Growth and Angiogenesis in Fischer
344 Rats1
Siqing Shan, A. Craig Lockhart, Wilfred Y. Saito, treated animals were >50% smaller than tumors in control
A. Merrill Knapp, Keith R. Laderoute, and animals. In addition, vascular length densities in peripheral
tumor zones were 30% less in treated compared with control
Mark W. Dewhirst2
animals. Together, these findings demonstrate that BTO-956
Department of Radiation Oncology, Duke University Medical Center,
can inhibit angiogenesis induced by a growth factor in the
Durham, North Carolina 27710 [S. S., A. C. L., W. Y. S., M. W. D.],
and Pharmaceutical Discovery Division, SRI International, Menlo rat cornea and in the peripheral area of implanted tumors,
Park, California 94025 [A. M. K., K. R. L.] where tumor angiogenesis is most active.
INTRODUCTION
ABSTRACT Angiogenesis, the formation of new vessels from existing
BTO-956 [methyl-3,5-diiodo-4-(4ⴕ-methoxyphenoxy)- vasculature, has a critical role in primary tumor growth, inva-
benzoate], a novel tubulin-binding drug and thyroid hor- sion, and metastasis (1). Tumor-associated angiogenesis is a
mone analogue, was originally found to inhibit human car- multistep process that is controlled by both positive and nega-
cinoma cell proliferation in vitro and to have potent growth tive factors as well as by complex interactions among tumor
delay activity in human breast and ovarian carcinoma xe- cells, host endothelium, stromal cells, and extracellular matrix
nografts in nude mice. Here we report that BTO-956 given components. Moreover, because tumor vascular networks are
to Fischer 344 rats also inhibits corneal angiogenesis and the formed within microenvironments intrinsic to the tumor mass,
growth and neovascularization of the R3230Ac rat mam- they have markedly different properties compared with those in
mary carcinoma tumor implanted in skin-fold window normal tissues. These tissue-level differences make angiogenic
chambers. Hydron pellets containing recombinant human tumor endothelial cells an attractive target for the development
basic fibroblast growth factor (50 ng) and Sucralfate (20 g) of new anticancer therapies. Current antivascular or antiangio-
were implanted into surgically created corneal micropockets genic approaches include treatment with cytotoxic agents hav-
(day 0). BTO-956 was administrated by oral gavage (500 ing some selectivity for proliferating vascular endothelial cells,
mg/kg, twice a day for 6 days) on days 1– 6 (controls received natural angiogenesis inhibitors, modified soluble receptors that
vehicle alone). On day 7, rats received retrogade infusions of interfere with angiogenic signal transduction, and synthetic
India ink via the thoracic aorta to visualize the corneal compounds capable of selectively inhibiting endothelial cell
vasculature. Digitized images of slide-mounted corneas from proliferation or remodeling of the extracellular matrix (2, 3).
control and treated animals were taken with a microscope. Some conventional anticancer drugs have antivascular or
For the tumor growth and angiogenesis study, small pieces antiangiogenic activity in addition to their cytotoxic effects
of R3230Ac tumor from a donor rat were implanted into toward tumor cells (4, 5). For example, tubulin-binding agents
surgically prepared window chambers (day 0). BTO-956 that cause mitotic arrest can have antivascular or antiangiogenic
was given during days 5–11, and images of the tumors and activity. Colchicine is a tubulin-binding agent that was reported
their vasculature were recorded on day 12. No body weight to produce hemorrhagic necrosis in experimental tumors (6). In
loss was observed in either study. BTO-956 significantly addition, colchicine was reported to be cytotoxic toward endo-
inhibited corneal angiogenesis (by 50 – 80%), as assessed by thelial cells in capillary sprouts (7). Combretastatin A4, another
measurements of limbal circumference displaying neovascu- tubulin-binding molecule isolated from the South African tree
larization, vessel length, vascularized area, and vascular Combretum caffrum, displays potent and selective toxicity to-
area density. In the window chamber assay, tumors from ward tumor vasculature and to perturb tumor perfusion and
energy status (8, 9). This latter toxicity is termed vascular
targeting.
BTO-956 [methyl-3,5-diiodo-4-(4⬘-methoxyphenoxy)ben-
Received 1/2/01; revised 5/14/01; accepted 5/15/01. zoate; Fig. 1] was originally developed as a thyroid hormone
The costs of publication of this article were defrayed in part by the analogue, but failed to show any physiological activity associ-
payment of page charges. This article must therefore be hereby marked ated with thyroid hormone metabolism (10). However, in recent
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact. studies we demonstrated that BTO-956 inhibits the proliferation
1
This research was supported by the California Breast Cancer Research of human carcinoma cells in vitro and that it has potent growth
Program (Grant 4CB-0032 to K. R. L.) and Large Scale Biology Cor- delay activity in human breast and ovarian carcinoma xenografts
poration, Vacaville, CA. in nude mice (11, 12). In addition, we found that the cytotoxicity
2
To whom requests for reprints should be addressed, at Department of
Radiation Oncology, Duke University Medical Center, Durham, NC
of BTO-956 toward tumor cells involves mitotic arrest associ-
27710-3455. Phone: (919) 684-4711; Fax: (919) 684-8718; E-mail: ated with disruption of cellular microtubule assembly that prob-
dewhirst@radonc.duke.edu. ably arises from binding of the drug to the colchicine site of
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.Clinical Cancer Research 2591
were used for this study. Animals were kept in temperature-
controlled rooms (24°C) on a 12-h light-dark cycle with access
to rat chow and tap water ad libitum. Following anesthesia with
sodium pentobarbital (Abbott Laboratories, North Chicago, IL)
given i.p. at 50 mg/kg of body weight, a rat was placed under a
Fig. 1 Structure of BTO-956. dissecting microscope, and a drop of Ophthaine was applied on
the cornea of one eye. The eye was proptosed by stretching
sutures on the upper and lower eyelids. A small superficial
tubulin (12). This finding was unexpected considering the struc- incision was made through the cornea center, and a micropocket
tural similarity of BTO-956 to thyroid hormone, and suggested was created by separating the lamella of the stroma toward the
that it represents a new and novel class of antitumor agent. limbus with a modified iris spatula. The distance between the
Because tubulin-binding drugs can have antiangiogenic activity, bottom of the micropocket and the limbus was ⬃1 mm. A
we hypothesized that BTO-956 could also inhibit angiogenesis. prepared sterile Hydron pellet was rehydrated with a drop of
Here we present findings showing that BTO-956 has strong sterile saline and placed into the corneal micropocket. The
antiproliferative activity toward human microvascular endothe- cornea was covered with gentamicin ophthalmic ointment (Alle-
lial cells in vitro and substantial antiangiogenic activity in vivo, ran, Inc., Irvine, CA) after surgery. Rats were randomly divided
as assessed by its effect on rat corneal neovascularization in- into treated and control groups.
duced by a growth factor (13) and on tumor angiogenesis in a Before administration, BTO-956 was suspended at 150
window chamber model of the R3230Ac rat mammary carci- mg/ml in 0.5% methylcellulose (Sigma) in saline containing
noma (14). 0.1% Tween 80 (Sigma). The drug was administrated by oral
gavage at a dose of 500 mg/kg of body weight twice a day for
MATERIALS AND METHODS 6 days beginning from the first postoperative day. The controls
received vehicle only. Body weights of all animals were mon-
BTO-956. BTO-956 used in this study is an experimental
itored and recorded daily. On the 7th postoperative day, animals
drug that was synthesized at SRI International by procedures
were anesthetized with sodium pentobarbital (50 mg/kg), and
published in the literature (12).
the thoracic aorta was rapidly cannulated in a retrograde fashion
Rat Corneal Neovascularization Assay. Hydron poly-
with PE 100 tubing. An incision was made in the right auricle
mer (IFN Sciences, New Brunswick, NJ) was dissolved in
for drainage of blood and saline during flushing, and 40 – 60 ml
absolute ethanol (12% w/v) in a rotator at 37°C overnight and
of saline was injected via the tubing to wash out the blood from
stored at room temperature before pellet making. A stock solu-
the vessels in the upper part of the body until the eyes, ears, and
tion of bFGF3 (recombinant human bFGF; R&D Systems, Inc.,
nose became pale. Ten ml of Higgins waterproof India ink
Minneapolis, MN) at a concentration of 200 ng/l was prepared
(Sanford, Bellwood, IL) were injected via the tubing to visualize
by dissolving the lyophilized product with sterile PBS contain-
corneal vessels. The eyes were enucleated and fixed in 10%
ing 0.1% BSA. Aliquots of the stock bFGF solution (5 or 10 l)
neutralized buffered formaldehyde and the corneas were dis-
were then prepared and stored at ⫺80°C. Sucralfate (sucrose
sected and mounted on slides. Slide-mounted corneal images
octasulfate aluminum complex; Sigma Chemical Co., St. Louis,
were taken using a Carl Zeiss MPS intravital microscope (Carl
MO) stock solution was prepared by suspending Sucralfate in
Zeiss, Hanover, MD) with a camera connected to a personal
sterile PBS at 100 g/l. This solution was stored at 4°C.
computer equipped with Scion Image software and a frame
Each pellet for the corneal pocket assay contained 50 ng of
grabber (Scion Corporation, Frederick, MD).
bFGF and 20 g of Sucralfate in 3 l of casting gel, which was
For the quantitative analysis of corneal neovascularization,
constituted as a 50:50 (v/v) mixture of Hydron gel and bFGF-
various parameters of the corneal images were measured by an
Sucralfate-PBS. The pellets were prepared the day before cor-
investigator who was unaware of the treatment assignment. The
neal surgery in a laminar flow hood under sterile conditions. For
shortest distance between the implanted pellet and the limbus
example, to make 20 pellets, 4 l of Sucralfate suspension was
was marked as the “mid-line” and was measured. The arc along
added to a 5-l bFGF aliquot. Sterile PBS (21 l) was then
the limbus for the entire vascularized area was drawn, and the
added to this vial and vortexed; 30 l of Hydron gel was then
length of the arc was measured (Fig. 2). The circumference of
added, followed by vigorous vortexing for 1 min. This casting
this area (i.e., the arc angle) was calculated by the following
gel was promptly pipetted in 3.0-l drops on a sterile Teflon
equation: Circumference (in clock hours) ⫽ Arc length ⫻
sheet (Small Parts, Inc., Miami Lakes, FL) in a Petri dish. The
360/D/30, where D is the corneal diameter, obtained by aver-
Petri dish was placed in a 4°C refrigerator overnight to allow for
aging diameters of corneal images at lower magnification from
polymerization. Dried discs of uniform size (⬃2 mm in diam-
20 rats. The marked arc was evenly divided into six sections.
eter) were chosen for use.
The perpendicular distances between vessel tips and the limbus
Female Fischer 344 rats (n ⫽ 16 animals; 8 –10 weeks of
at the five intersection points of the arc were measured, and the
age; approximate weight, 150 g; Charles River, Raleigh, NC)
average vessel length was calculated. The perpendicular dis-
tance between the tip of the longest vessel and the limbus
(longest vessel length) was also measured. To measure the
3
The abbreviations used are: bFGF, basic fibroblast growth factor;
percentage of vascular area (i.e., the vascular area density), three
HMVEC, human microvascular endothelial cell; VEGF, vascular endo- circles with a diameter of 100 pixels were located between the
thelial growth factor. pellet and the limbus. The central circle was on the mid-line, the
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.2592 BTO-956 Inhibits Tumor Angiogenesis
implant. Similarly, vascular morphology was scored as follows:
1, scant few blood vessels, especially around the tumor implant;
2, moderately abundant blood vessels; and 3, abundant blood
vessels or newly formed vessels adjacent to the implant. For
evaluating blood flow, the following scores were used: 1, less
than one-third of the visible blood vessels had active flow
around the implant; 2, functioning vessels were observed in
more than one-third but less than two-thirds of the total vessels;
and 3, more than two-thirds of the vessels were functioning with
brisk blood flow. Only those windows with ⬎6 points on this
scale were included for further study. In many prior studies in
Fig. 2 Method for quantitation of corneal vascularization. The arc untreated animals, we have found that this selection procedure
(Arc) along the limbus for the vascularized area was drawn, and its
length was measured for calculation of the circumference of the neo-
effectively eliminates chamber preparations that fail to grow
vascularized area (i.e., the arc angle). The marked arc was evenly tumors for various technical reasons.
divided into six sections, distances between vessel tips and the limbus at Tumor-window-bearing animals were randomly divided
the five intersection points of the arc were measured, and the average into treated and control groups, stratified by the window scores.
vessel length was calculated. The perpendicular distance between the tip Treatment with BTO-956 was performed from the 5th to the
of the longest vessel and the limbus (longest vessel length, L) was also
measured. Three circles (C) indicate fields for measuring the vascular 11th postoperative day at the same doses as those described
area density. The area enclosed by the dotted line and the arc was above for the corneal neovascularization assay. On day 12, the
measured and presented as total vascularized area. (See “Materials and tumor windows were evaluated for the effect of the drug on
Methods” for details). tumor growth and vascularization. Tumor areas were measured
with lower magnification images (objective, ⫻5 or ⫻2.5, de-
pending on tumor size) of whole tumors. Tumor vasculature was
evaluated based on four peripheral tumor areas, three to four
other two lateral circles were symmetrically placed in each side central tumor areas (depending on tumor size), and four areas in
of the central one in the second and the fifth sections, respec- the surrounding granulating tissue, using higher magnification
tively. The total area (pixel numbers) of each circle (Area-T) (objective, ⫻20). Measurements were made by an investigator
and the area for vessels in each circle (total number of black who was unaware of the treatment protocol. Image analysis
pixels at threshold setting) were measured (Area-V). The vas- software, combined with observation of videotaped images
cular area density was defined as the ratio of Area-V to Area-T. showing blood flow, was used to measure the cumulative length
An average value from these three circles was obtained for each of all vessels in focus in each image. The vascular length density
cornea. Total vascularized corneal area was also measured by was calculated by dividing the total vessel length in a frame by
marking the perimeter of the vascularized area with image the area of the frame. The diameters of all vessels in each image
analysis software. Images were calibrated against a stage mi- at high magnification were also measured and averaged. All
crometer image taken at the same magnification and zoom measurements were calibrated against micrometer images at the
parameters. same magnification.
Dorsal Skin-Fold Window Chamber Assay. Details of Effect of BTO-956 on Proliferation of R3230Ac Rat
the design and the surgical technique used for the rat dorsal Mammary Carcinoma and HMVECs. To investigate the
skin-fold window chamber assay have been described elsewhere effect of BTO-956 on proliferation of R3230Ac cells, cells were
(14). Briefly, the anatomical midline of the dorsal skin flap of an first plated at 106 cells/100-mm-diameter plastic culture dish in
anesthetized rat was sutured to a pair of C clamps, and the DMEM containing 10% fetal bovine serum (Sigma) and 25 mM
skin-fold was retracted from the body surface by hanging the C HEPES (pH 7.4), and incubated at 37°C in 5% CO2 for 2 days.
clamp on a plastic surgical stage. A 1-cm circle of skin on each Cells were then plated in a 96-well culture plate at 2 ⫻ 103
side of the skin-fold was surgically removed, leaving two layers cells/well in 100 l of DMEM-HEPES-10% fetal bovine serum
of fascia containing a few microvessels. After a pair of window and incubated at 37°C overnight. Normal HMVECs isolated
frames were mounted on both sides of the flap, a piece of a from dermis were obtained from Clonetics Corporation (Walk-
R3230Ac rat mammary carcinoma (⬃0.5 mm3) from a donor rat ersville, MD) and cultured on 100-mm-diameter plastic culture
was implanted onto the fascia in the window and the chamber dishes in EGM-2-MV growth medium according to the suppli-
was sealed with glass coverslips. On the 4th postoperative day, er’s instructions. HMVECs were incubated in a 5% CO2-air
all tumor-containing windows were evaluated using an intravital atmosphere at 37°C until they were 70 –90% confluent, and then
microscope equipped with a video camera. Two investigators were plated in a 96-well culture plate at 5 ⫻ 103 cells/well in
observed live images from the camera monitor and crudely 100 l of EGM-2-MV and incubated at 37°C overnight. BTO-
scored the viability of the tumor implants in each window 956 was added to both cell types over the concentration range
chamber, with 1 to 3 points assigned to the categories of size, 0 –10 M; cells were then incubated at 37°C for 3 days. The
vascular morphology, and blood flow. Thus, by drawing implant effect of BTO-956 on cell proliferation was measured on the 4th
perimeters for images of individual window chambers on a day using the nontoxic redox-sensitive dye Alamar Blue in a
transparency sheet in front of the monitor, implant sizes were colorimetric assay.
compared and scored as follows: 1, small implant, or an implant Statistical Analysis. All data were reported as the
with visible necrotic areas; 2, medium implant; and 3, large mean ⫾ SE for each group. The statistical significance of
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.Clinical Cancer Research 2593
Fig. 4 Quantitative comparison of corneal neovascularization induced
by bFGF in Fischer 344 rats. All parameters associated with corneal
neovascularization were significantly lower in BTO-956-treated animals
(䡺) compared with controls (f). The circumference values (Clock
Hours) for corneal neovascularization in treated and control animals
were 1.7 ⫾ 0.1 (n ⫽ 8) and 3.1 ⫾ 0.3 (n ⫽ 8), respectively (A). The
average vessel lengths in treated and control animals were 0.4 ⫾ 0.1 mm
(n ⫽ 8) and 0.8 ⫾ 0.1 mm (n ⫽ 8), respectively. The maximum vessel
Fig. 3 Typical images of corneal neovascularization induced by bFGF. lengths in treated and control animals were 0.8 ⫾ 0.1 mm (n ⫽ 8) and
A polymer pellet containing 50 ng of bFGF was implanted into a 1.2 ⫾ 0.1 mm (n ⫽ 8), respectively (B). The vascular area densities in
surgically created corneal micropocket (the oval dark area) in one eye treated and control animals were 0.04 ⫾ 0.01 (n ⫽ 8) and 0.24 ⫾ 0.04
of a Fischer 344 rat. BTO-956 (500 mg/kg) was administered p.o. twice (n ⫽ 8), respectively (C). The total vascular areas in treated and control
a day during postoperative days 1– 6. Control animals received vehicle animals were 1.5 ⫾ 0.3 mm2 (n ⫽ 8) and 4.3 ⫾ 0.6 mm2 (n ⫽ 8),
only. On day 7, the corneal neovasculature was visualized by ink respectively (D). Bars, SE. ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.
angiography (for details, see “Materials and Methods”). In control
animals, abundant new vessels grew from the limbus toward the pellet
(A), whereas newly formed vessels in the corneas of rats treated with
BTO-956 were scarce (B).
eters relevant for measuring the extent of corneal neovascular-
ization were significantly lower in BTO-956-treated compared
with control animals. The circumference of neovascularization
differences between treated and control groups was determined in treated rat corneas was reduced by 45% compared with
using an unpaired t test or the Mann-Whitney U test. Statisti- controls (P ⬍ 0.01; Fig. 4A). Average and maximum vessel
cally significant differences were defined as having a P ⬍ 0.05. lengths were reduced by 50% (P ⬍ 0.001) and 38% (P ⬍ 0.01;
Fig. 4B), respectively, and the vascular area density and the total
RESULTS vascular area were decreased by 84.5% (P ⬍ 0.01; Fig. 4C) and
No significant body weight loss or behavioral changes 65% (P ⬍ 0.01; Fig. 4D), respectively, compared with controls.
were found for any animals treated with BTO-956 in either BTO-956 Inhibits Tumor Growth and Angiogenesis.
study. This result concerning the safety of BTO-956 is consist- Tumors growing in windows of BTO-956-treated animals were
ent with those of previous in vivo studies showing that the drug much smaller than those in controls (Fig. 5). The treated tumors
is well tolerated by animals given similar doses administered were also less vascularized, especially in the periphery where
orally (12). angiogenesis is prominent. Specifically, tumor areas were re-
BTO-956 Inhibits Corneal Angiogenesis. The pellet duced ⬎50% in BTO-956-treated compared with control ani-
sizes and the distances between the pellet and the limbus for mals: 23.8 ⫾ 2.5 mm2 (n ⫽ 8) for control animals versus 10.8 ⫾
each group were similar (data not shown). Corneal neovascu- 3.4 mm2 (n ⫽ 9) for treated animals (P ⬍ 0.05; Fig. 6A).
larization induced by bFGF in BTO-956-treated animals was Vascular length density in peripheral tumor areas in BTO-956-
markedly reduced compared with vehicle-treated control ani- treated animals was significantly less than that of the controls:
mals (Fig. 3). Quantitative comparison showed that all param- 5.9 ⫾ 0.8 mm/mm2 (n ⫽ 9) for treated animals versus 7.8 ⫾ 0.6
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.2594 BTO-956 Inhibits Tumor Angiogenesis
Fig. 6 Quantitative comparison of the effect of BTO-956 on the
growth and neovascularization of the R3230Ac rat mammary carcinoma
implanted in rat dorsal skin-fold window chambers in Fischer 344 rats.
The average sizes of tumors in BTO-956-treated and control animals, as
estimated by measurements of two-dimensional areas, were 10.8 ⫾ 3.4
mm2 (n ⫽ 8) and 23.8 ⫾ 2.5 mm2 (n ⫽ 9), respectively (A). The
vascular length densities in the peripheral tumor zones in treated and
control animals were 5.9 ⫾ 0.8 mm/mm2 (n ⫽ 9) and 7.8 ⫾ 0.6
Fig. 5 Inhibition of tumor growth and angiogenesis in the rat dorsal mm/mm2 (n ⫽ 8), respectively (B). Bars, SE. ⴱ, P ⬍ 0.05.
skin-fold window chamber by orally administered BTO-956. R3230Ac
rat mammary carcinoma tumors in window chambers were exposed to
BTO-956 or vehicle beginning on day 5 postimplantation for a total of
7 days (for details, see “Materials and Methods”). On day 12, images of
the window chambers were taken and analyzed. Tumor area was meas-
Effect of BTO-956 on Proliferation of R3230Ac Tumor
ured at lower magnification (objective, ⫻2.5 or ⫻5, depending on the
tumor size). Measurements were calibrated against micrometer images Cells and HMVECs in Vitro. Fig. 7 shows that BTO-956
at the same magnification. A, representative large tumor with abundant inhibited the in vitro proliferation of R3230Ac cells with an IC50
vascularization in a control animal (⫻2.5). B, representative tumor in an (the concentration required to decrease the number of cells by
animal treated with BTO-956, showing a smaller size and less vascu- 50% relative to the control) of ⬃400 nM. Using the same in vitro
larization (⫻5), compared with the control image. Arrowheads indicate
tumor margins. Bars, 800 m. assay, we found that BTO-956 also inhibited the proliferation of
HMVECs with an IC50 of ⬃200 nM (Fig. 7). Therefore, con-
sistent with other findings reported for tubulin-binding drugs,
BTO-956 can inhibit the proliferation of both tumor and normal
mm/mm2 (n ⫽ 8) for controls (P ⬍ 0.05; Fig. 6B). In contrast, microvascular endothelial cells.
there was no significant difference in the vascular length density
between treated and control groups in central tumor areas [3.3 ⫾ DISCUSSION
0.8 mm/mm2 (n ⫽ 9) for treated animals versus 4.2 ⫾ 0.2 The major findings of this study are that oral administration
mm/mm2 (n ⫽ 8) for controls] or in surrounding granulating of the tubulin-binding drug BTO-956 inhibited (a) bFGF-
tissue [8.7 ⫾ 1.1 mm/mm2 (n ⫽ 9) for treated animals versus induced rat corneal neovascularization and (b) tumor growth
8.7 ⫾ 0.8 mm/mm2 (n ⫽ 8) for controls]. Vascular diameter in and angiogenesis of the R3230Ac rat mammary carcinoma. The
the central tumor areas was larger than that in peripheral tumor antiangiogenic activity of BTO-956 was detectable in the pe-
areas in both groups. However, there were no significant differ- ripheral areas of these implanted tumors, where the most active
ences in vascular diameter between the two groups in the angiogenesis occurs (15, 16). These effects of BTO-956 are
peripheral tumor areas [32.2 ⫾ 4.5 m (n ⫽ 9) for treated consistent with a growing body of literature suggesting that
animals versus 36.2 ⫾ 5.3 m (n ⫽ 8) for controls], central small-molecule drugs having the ability to disrupt cellular mi-
tumor areas [35.8 ⫾ 9.6 m (n ⫽ 9) for treated animals versus crotubule dynamics can be potent antivascular or antiangiogenic
32.0 ⫾ 4.4 m (n ⫽ 8) for controls], or surrounding areas agents (e.g., see Refs. 17, 18). The molecular mechanism of this
[21.5 ⫾ 3.3 m (n ⫽ 9) for treated animals versus 19.2 ⫾ 1.4 antiangiogenic effect of BTO-956 is unknown at present. Be-
m (n ⫽ 8) for controls]. cause BTO-956 can strongly inhibit the proliferation of both
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.Clinical Cancer Research 2595
antiproliferative response of vascular endothelial cells to BTO-
956 is an important area for further research.
The results from the tumor window chamber assay for the
R3230Ac rat mammary carcinoma provide further demonstra-
tion that BTO-956 inhibits tumor growth by at least 50%, as
assessed by area, compared with controls. Indeed, if the volumes
of these tumors were considered, this antitumor effect of BTO-
956 would be significantly greater. Other studies have demon-
strated that the angiogenic activity in experimental tumors is
greatest in peripheral zones (15, 16). Consistent with this con-
clusion, the results reported here show that the vascular length
density in the periphery of the R3230Ac tumor was significantly
decreased in the window chambers of BTO-956-treated animals,
Fig. 7 Effect of BTO-956 on the proliferation of R3230Ac rat mam- whereas the vascular length density in central areas of these
mary carcinoma cells and normal HMVECs in vitro. Percentage of total tumors was unchanged. These findings indicate that the antian-
cells (Percent of Total Cells) ⫽ (number of cells for a treatment giogenic effect of BTO-956 is most potent in actively angio-
concentration ⫺ number of cells remaining after the most effective genic areas of a tumor.
concentration)/(number of untreated or control cells ⫺ number of cells
remaining after the most effective concentration) ⫻ 100. Bars, SD. The in vitro study of the effect of BTO-956 on R3230Ac
tumor cell proliferation showed cytotoxic or cytostatic activity
similar to that described for human breast and ovarian cancer
cell lines (11, 12). Therefore, taken with the antiangiogenic
R3230Ac tumor cells and normal microvascular endothelial effect of BTO-956 detected in the window chamber assay, we
cells in vitro, it is likely that its ability to inhibit both corneal and suggest that the overall effect of the drug on the growth of the
tumor angiogenesis involves an effect on endothelial cell pro- R3230Ac tumor was the result of cytotoxicity toward both
liferation and survival. As reported in our previous study (12), angiogenic microvascular endothelial cells and the tumor cells
BTO-956 can arrest proliferating tumor cells at a G2-M check- themselves. Finally, as found in our earlier in vivo studies of the
point and cause apoptosis, similar to the effect of other tubulin- effect of BTO-956 on tumor growth (12), we observed no
binding agents such as colchicine, vinblastine, and paclitaxel. serious toxic effects of the drug on tumor-bearing animals, such
BTO-956 has a structure similar to that of combretastatin A-4 as weight loss or behavioral abnormalities, even at p.o.-admin-
(12), and both of these molecules apparently bind to colchicine istered doses as high as 500 mg/kg/day given daily for up to 7
sites on -tubulin. However, whereas combretastatin A-4 has days. Further toxicological studies will be necessary to fully
been shown to exert a direct and rapid antivascular effect on evaluate whether BTO-956 has significantly deleterious effects
tumor microvessels (8, 9), the findings from the corneal neo- on normal tissues in the rat. Previous pharmacokinetic studies
vascularization assay reported here suggest that BTO-956 is showed that human breast tumor xenografts contained an aver-
more likely to function as an antiangiogenic rather than an age of ⬃9 g-equivalents of p.o.-administered BTO-956 per g
antivascular agent. In terms of proangiogenic factors in of tumor tissue after oral administration of 0.5 g/kg twice daily
R3230Ac tumors, we have shown by immunohistochemistry for 3 days (12). Therefore, because much of the drug was not
that those tumors express both bFGF and VEGF.4 We have also bioavailable to these tumor-bearing mice and was excreted, its
found that blockade of VEGF signaling with a soluble VEGF effect on tumor growth involved only a small fraction of the
receptor protein blocks neovascularization in these tumors (19) administered dose. Together with the possibility that BTO-956
and have shown that SU-5416, a selective inhibitor of VEGF may have more than one cellular target mediating both its
receptor Flk-1, inhibits R3230Ac tumor growth and angiogen- cytotoxicity and effect on tumor growth, the pharmacokinetic
esis.5 However, because BTO-956 is a tubulin-binding drug, studies provide an explanation for the ability of rodents to
presumably its antiangiogenic effect at least partly involves tolerate large doses of the drug. These findings indicate that this
disruption of cellular microtubule networks (12), although it has drug can be administrated p.o. with considerable safety and
not been established that -tubulin is the actual in vivo target of suggest that it has promise for eventual clinical applications.
the drug. In this connection, recent subcellular fractionation These applications may require further research concerning for-
studies have shown that on exposure of R3230Ac cultures to mulations to increase the bioavailability of BTO-956 or its
[14C]BTO-956, approximately twice as much label accumulated derivatives.
in the plasma membrane fraction compared with the soluble/ In this study, we used vascular length density, as measured
cytosolic fraction.6 Therefore, BTO-956 may have more than in skin-fold window chamber tumors to assess the antiangio-
one cellular target that could mediate its cytotoxic effect on genic activity of this drug during tumor growth. This method is
proliferating cells. Establishing the downstream effectors of the well established as an assay for this end point, as has been
reported by others and us (19 –23). The difference between this
method and simple measurements of microvessel density from
histological sections is that the method reflects changes in
4
Dewhirst et al., unpublished data.
functional vasculature. In this case, we define functional vascu-
5
Shan et al., unpublished data. lature as reflecting those vessels that are perfused with red cells.
6
Laderoute et al., unpublished results. Because the nutrients that are necessary for tumor growth must
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.2596 BTO-956 Inhibits Tumor Angiogenesis
come from functional vasculature, this method more robustly 12. Chen, X., Pine, P., Knapp, A. M., Tusé, D., and Laderoute, K. R.
reflects the physiological effect of an antiangiogenic compound Oncocidin A1: a novel tubulin-binding drug with antitumor activity
on tumor growth, which is also measured in the same experi- against human breast and ovarian carcinoma xenografts in nude mice.
Biochem. Pharmacol., 56: 623– 633, 1998.
ment. The process of metastasis also requires tumor cell inva-
sion into functional vasculature. One potential disadvantage of 13. Polverini, P. J., Bouck, N. P., and Rastinejad, F. Assay and purifi-
cation of naturally occurring inhibitor of angiogenesis. Methods Enzy-
this method is that we are only able to measure vasculature on mol., 198: 440 – 450, 1991.
one surface of the preparation when we use transillumination. It
14. Pappenfuss, D., Gross, J. F., Intaglietta, M., and Treese, F. A. A
is possible that this measurement does not reflect what is oc- transparent access chamber for the rat dorsal skin fold. Microvasc. Res.,
curring in deeper layers of the tumor. However, this is unlikely 18: 311–318, 1979.
because the surface of the tumor represents that region of tumor 15. Fukumura, D., Xavier, R., Sugiura, T., Chen, Y., Park, E. C., Lu, N.,
with most active angiogenesis (15, 16). Selig, M., Nielsen, G., Taksir, T., Jain, R. K., and Seed, B. Tumor
Another potential limitation of this method is that mam- induction of VEGF promoter activity in stromal cells. Cell, 94: 715–
mary tumors are grown in an ectopic site. It is well established 725, 1998.
that tumor growth and angiogenesis are affected by the site of 16. Li, C. Y., Shan, S., Huang, Q., Braun, R. D., Lanzen, J., Hu, K., Lin,
transplant (24 –26). Thus, the degree of angiogenesis inhibition P., and Dewhirst, M. W. Initial stages of tumor cell-induced angiogen-
esis: evaluation via skin window chambers in rodent models. J. Natl.
found in this model with BTO-956 might be different from that
Cancer Inst. (Bethesda), 92: 143–147, 2000.
seen if the tumors were grown in their orthotopic sites. Addi-
17. Iyer, S., Chaplin, D. J., Rosenthal, D. S., Boulares, A. H., Li, L. Y.,
tional studies would have to be done to determine the magnitude and Smulson, M. E. Induction of apoptosis in proliferating human
of such an effect, but are outside the scope of the present study. endothelial cells by the tumor-specific antiangiogenesis agent combret-
astatin A-4. Cancer Res., 58: 4510 – 4514, 1998.
REFERENCES 18. Suzuki, M., Okano, A., Tsuji, T., Akiyama, Y., Tsuruo, T., Saito, S.,
Hori, K., and Sato, Y. Evaluation of antivascular and antimitotic effects
1. Folkman, J. Clinical application of research on angiogenesis. Semi-
nars in Medicine of the Beth Israel Hospital, Boston. N. Engl. J. Med., of tubulin binding agents in solid tumor therapy. Jpn. J. Cancer Res., 90:
333: 1757–1763, 1995. 1387–1395, 1999.
2. Gasparini, G. The rationale and future potential of angiogenesis 19. Lin, P., Sankar, S., Shan, S., Dewhirst, M. W., Polverini, P. J.,
inhibitors in neoplasia. Drugs, 58: 17–38, 1999. Quinn, T. Q., and Peters, K. G., Inhibition of tumor growth by targeting
tumor endothelium using a soluble vascular endothelial growth factor.
3. Arap, W., Pasqualini, R., and Ruoslahti, E. Chemotherapy targeted to
Cell Growth Differ., 9: 49 –58, 1998.
tumor vasculature. Curr. Opin. Oncol., 10: 560 –565, 1998.
20. Lin, P., Polverini, P., Dewhirst, M., Shan, S., Rao, P. S., and Peters,
4. Presta, M., Rusnati, M., Belleri, M., Morbidelli, L., Ziche, M., and
K. Inhibition of tumor angiogenesis using a soluble receptor establishes
Ribatti, D. Purine analogue 6-methylmercaptopurine riboside inhibits
early and late phases of the angiogenesis process. Cancer Res., 59: a role for Tie2 in pathologic vascular growth. J. Clin. Investig., 100:
2417–2424, 1999. 2072–2078, 1997.
5. Ferrante, K., Winnograd, B., and Canetta, R. Promising new devel- 21. Jain, R. K., Safabakhsh, N., Sckell, A., Chen, Y., Jiang, P., Benja-
opments in cancer chemotherapy. Cancer Chemother. Pharmacol., 43 min, L., Yuan, F., and Keshet, E. Endothelial cell death, angiogenesis,
(Suppl.): S61–S68, 1999. and microvascular function after castration in an androgen-dependent
tumor: role of vascular endothelial growth factor. Proc. Natl. Acad. Sci.
6. Ludford, R. J. The action of toxic substances upon the division of
USA, 95: 10820 –10825, 1998.
normal and malignant cells in vitro and in vivo. Arch. Exp. Zellforsch.,
18: 411– 441, 1936. 22. Haroon, Z. A., Hettasch, J. M., Lai, T. S., Dewhirst, M. W., and
Greenberg, C. S. Tissue transglutaminase is expressed, active, and
7. Ludford, R. J. Colchicine in the experimental chemotherapy of
cancer. J. Natl. Cancer Inst. (Bethesda), 6: 89 –101, 1945. directly involved in rat dermal wound healing and angiogenesis. FASEB
J., 13: 1787–1795, 1999.
8. Dark, G. G., Hill, S. A., Prise, V. E., Tozer, G. M., Pettit, G. R., and
Chaplin, D. J. Combretastatin A-4, an agent that displays potent and 23. Haroon, Z. A., Lai, T. S., Hettasch, J. M., Lindberg, R. A., Dew-
selective toxicity toward tumor vasculature. Cancer Res., 57: 1829 – hirst, M. W., and Greenberg, C. S. Tissue response to tumor invasion
1834, 1997. and inhibits tumor growth. Lab. Investig., 79: 1679 –1686, 1999.
9. Beauregard, D. A., Thelwall, P. E., Chaplin, D. J., Hill, S. A., Adams, 24. Takahashi, Y., Ellis, L. M., Wilson, M. R., Bucana, C. D., Kitadai,
G. E., and Brindle, K. M. Magnetic resonance imaging and spectroscopy Y., and Fidler, I. J. Progressive upregulation of metastasis-related genes
of combretastatin A4 prodrug-induced disruption of tumor perfusion and in human colon cancer cells implanted into the cecum of nude mice.
energetic status. Br. J. Cancer, 77: 1761–1767, 1998. Oncol Res., 8: 163–169, 1996.
10. Stasilli, N. R., Kroc, R. L., and Meltzer, R. I. Antigoitrogenic and 25. Pettaway, C. A., Pathak, S., Greene, G., Ramirez, E., Wilson, M. R.,
calorigenic activities of thyroxine analogues in rats. Endocrinology, 64: Killion, J. J., and Fidler, I. J. Selection of highly metastatic variants of
62– 82, 1959. different human prostatic carcinomas using orthotopic implantation in
11. Kun, E., Mendeleyev, J., Bauer, P. I., Kirsten, E., Zhen, J., Young, nude mice. Clin. Cancer Res., 2: 1627–1636, 1996.
L. T., Vidair, C. A., and Pine, P. Induction of tumor apoptosis by 26. Fukumura, D., Yuan, F., Monsky, W. L., Chen, Y., and Jain, R. K.
methyl-3,5-diiodo-4-(4⬘-methoxyphenoxy)benzoate (DIME). Int. J. On- Effect of host microenvironment on the microcirculation of human
col., 9 (Suppl.): 829, 1996. colon adenocarcinoma. Am. J. Pathol., 151: 679 – 688, 1997.
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.The Novel Tubulin-binding Drug BTO-956 Inhibits R3230Ac
Mammary Carcinoma Growth and Angiogenesis in Fischer 344
Rats
Siqing Shan, A. Craig Lockhart, Wilfred Y. Saito, et al.
Clin Cancer Res 2001;7:2590-2596.
Updated version Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/7/8/2590
Cited articles This article cites 22 articles, 8 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/7/8/2590.full.html#ref-list-1
Citing articles This article has been cited by 3 HighWire-hosted articles. Access the articles at:
http://clincancerres.aacrjournals.org/content/7/8/2590.full.html#related-urls
E-mail alerts Sign up to receive free email-alerts related to this article or journal.
Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.
Permissions To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@aacr.org.
Downloaded from clincancerres.aacrjournals.org on November 8, 2015. © 2001 American Association for Cancer
Research.You can also read
